Effect of Erjing Pills on alleviating neuroinflammation of AD rats based on TLR4/NF-κB/NLRP3 pathway and its mechanism / 中国中药杂志

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aβ_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aβ_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aβ_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aβ_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1β, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aβ_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1β, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aβ and expression of p-Tau, thereby restoring the hippocampal morphological structure.

Mechanism analysis of repeatedly steamed and sundried Rehmanniae Radix Praeparata in delaying brain aging in ovariectomized mice based on proteomics / 中国中药杂志

The present study explored the effect and mechanism of repeatedly steamed and sundried Rehmanniae Radix Praeparata(RRP) in delaying brain aging in ovariectomized mice. After ovariectomy, the mice were randomly divided into a model group, an estradiol valerate group(0.3 mg·kg~(-1)), and low-(1.0 g·kg~(-1)), medium-(2.0 g·kg~(-1)), and high-dose(4.0 g·kg~(-1)) RRP groups, and a sham operation group was also set up, with 15 mice in each group. One week after the operation, intragastric administration was carried out for 15 consecutive weeks. The step-down test and Morris water maze test were used to detect the behavioral changes of mice. HE staining and Nissl staining were used to observe the morphological changes of mouse brain tissues. Immunohistochemistry was used to detect the expression of Aβ and ER_β in mouse brain tissues. The serum estrogen levels and cholinesterase and cholinesterase transferase levels in brain tissues of mice were detected by assay kits. The extracted hippocampal protein was detected by the Nano-ESI-LC-MS system, identified by the Protein Discovery, and analyzed quantitatively and qualitatively by the SIEVE. The PANTHER Classification System was used for GO analysis and KEGG pathway enrichment analysis of the differential proteins. Compared with the sham operation group, the model group showed decreased learning and memory ability, shortened step-down latency(P<0.05), prolonged escape latency(P<0.05), reduced platform crossings and residence time in the target quadrant, scattered nerve cells in the hippocampus with enlarged intercellular space, increased expression of Aβ-positive cells(P<0.05), declining expression of ER_β-positive cells and estrogen level(P<0.05), and weakened cholinergic function(P<0.05). Compared with the model group, the RRP groups showed improved learning and memory ability, prolonged step-down latency(P<0.05), increased estrogen level(P<0.05), neatly arranged nerve cells in the hippocampus with complete morphology, declining Aβ-positive cells, and elevated expression of ER_β-positive cells. A total of 146 differential proteins were screened out by proteomics, and KEGG pathway enrichment yielded 75 signaling pathways. The number of proteins involved in the dopaminergic synapse signaling pathway was the largest, with 13 proteins involved. In summary, RRP can delay brain aging presumedly by increasing the level of estrogen, mediating the dopaminergic synapse signaling pathway, and improving cholinergic function.

Effect of Erjingwan on Hippocampal Proteomics in AD Rats with Kidney Yin Deficiency Induced by Ovariectomy+ D-Galactose Combined with Aβ1-40 / 中国实验方剂学杂志

Objective::To explore the effect of Erjingwan on the biological basis of kidney yin deficiency Alzheimer' s disease(AD)rats induced by ovariectomy+ D-galactose combined with amyloid beta1-40 (Aβ1-40). Method::After ovariectomy, rats were randomly divided into five groups: model group, positive group, Erjingwan high, medium and low dose group, 11 rats in each group, and 11 rats in sham operation group. One week after operation, D-galactose was injected intraperitoneally for 7 weeks, and four weeks after operation, Aβ1-40 was injected unilaterally into hippocampus. The rats in model group and sham-operation group were given saline by intragastric administration 3 weeks after operation. The rats in high, middle and low dose groups of Erjingwan were given corresponding concentration (9.0, 4.5, 2.25 g·kg-1). The rats in positive control group were given Donepezil 1.0 mg·kg-1 with dosage of 10 mL·kg-1 once a day for 35 consecutive days. After 30 days of administration, the learning ability of the rats were examined using a Y-maze. The hippocampus tissues of rats were isolated. The morphology of hippocampus was observed by Nissl staining.The proteins were detected by Nanol-ESI liquid-mass spectrometry system and identified by protein Discovery software. Relative quantitative and qualitative analysis of differential proteins in hippocampus was performed by SIEVE software, and Gene Ontology of differential protein was performed by PANTHER Classification System database. String analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were performed on the differential proteins. Result::Compared with model group, the correct rate of Y maze in the high and middle dose groups of Erjingwan was significantly raised(P<0.05), and the number of neurons in the hippocampal CA1 area was significantly increased(P<0.01).115 differential proteins (Ratio>1.5 or Ratio<0.5) such as Insulin-like growth factor 1 receptors(IGF-1R) were found in the high-dose group of the Erjingwan group as well as 94 differential proteins such as Synaptophysin expressed in the middle-dose group of the Erjingwan. And there are 87 differential proteins such as Acetyl-CoA acetyltransferase-cytosolic in the positive drug group. It showed that these proteins were mainly divided into tubule-related proteins, heat shock proteins, energy metabolism-related proteins and AD-related proteins with GO analysis. It was found that the above differential proteins involved 93 signaling pathways such as Dopaminergic synaps by KEGG analysis. Conclusion::Erjingwan can improve cognitive impairment and neuronal damage in rats with kidney yin deficiency, possibly by altering the expression of multiple pathway-associated proteins such as phosphatidylinositol 3-kinase/protein kinase B signaling pathway(PI3K/Akt), insulin signaling pathway, and adenylate-activated protein kinase (AMPK)signaling pathway, estrogen signaling pathway, and Dopaminergic synapse.